DESCRIPTION: Diabetic retinopathy (DR) is the most common diabetic complication developing in both type1 and type2 diabetic patients and insofar is incurable. Hyperactivity of the renin angiotensin system (RAS), resulting in elevated concentrations of Angiotensin II (Ang II), is central to pathways leading to increased vascular inflammation, oxidative stress and endothelial dysfunction in diabetes and its associated complications including DR. The discovery of ACE2 established the existence of a protective axis of the RAS as a key regulator counteracting the deleterious effects of Ang II. We have recently demonstrated that increased expression of ACE2 and Ang1-7, the two key members of the protective axis, via AAV-mediated retinal gene delivery reduced diabetes-induced retinal pathology, validating that enhancing the ACE2/Ang1-7/MAS axis is a promising approach for treating patients with DR. In addition, enhancing Ang1-7 and ACE2 also improves lipid and glucose metabolism, ameliorates insulin resistance and confers protection against a variety of pathological conditions including diabetes-induced nephropathy and cardiovascular dysfunction. Thus an ideal strategy would be to enhance ACE2/Ang1-7 both systemically and locally at target tissues. This proposal takes advantage of an innovative concept based on the use of plant chloroplasts as highly efficient bioreactors for production and oral delivery of therapeutic proteins bio encapsulated in plant cells. The therapeutic protein is fused to chorea toxin subunit B, which not only facilitate the transmucosal transport into circulation, but also enhance tissue cell uptake by GM1 receptor mediated endocytosis. We have recently shown that orally delivered therapeutic proteins are widely distributed in organs including brain and retina crossing blood brain/retina barriers; oral delivery of bio encapsulated myelin basic protein ameliorated amyloid burden in advanced human and mouse Alzheimer's disease brains and retinas, prevented loss of retinal ganglion cells. Thus this novel concept provides a more efficient, low cost production and patient-friendly delivery of therapeutic proteins, particularly to the central nervous system, which has long been a major challenge. The goals of this proposal are to evaluate the efficacy of oral delivery of Ang1- 7 and ACE2 bio encapsulated in plant cells in prevention and treatment of DR in suitable animal models and to generate and characterize transplastomic ACE2 and Ang1-7 expression in edible lettuce in order to advance bio encapsulated protein delivery approach towards clinical investigations. Result from these proposed studies will provide proof-of-concept on feasibility and efficacy in animal models of DR using oral delivery of plant chloroplast- based therapeutic proteins, and set the foundation for initiating clinical trials by oral delivery of bio encapsulate ACE2 and Ang1-7 for prevention and treatment of DR.